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CD8 T cells contribute to protective immunity against Mycobacterium tuberculosis. In humans, M. tuberculosis reactive CD8 T cells typically recognize peptides associated to classical MHC class Ia molecules, but little information is available on CD8 T cells recognizing M. tuberculosis Ags presented by nonclassical MHC class Ib molecules. We show here that CD8 T cells from tuberculosis (TB) patients recognize HLA-E-binding M. tuberculosis peptides in a CD3/TCR αβ mediated and CD8-dependent manner, and represent an additional type of effector cells playing a role in immune response to M. tuberculosis during active infection. HLA-E-restricted recognition of M. tuberculosis peptides is detectable by a significant enhanced ex vivo frequency of tetramer-specific circulating CD8 T cells during active TB. These CD8 T cells produce type 2 cytokines upon antigenic in vitro stimulation, help B cells for Ab production, and mediate limited TRAIL-dependent cytolytic and microbicidal activity toward M. tuberculosis infected target cells. Our results, together with the finding that HLA-E/M. tuberculosis peptide specific CD8 T cells are detected in TB patients with or without HIV coinfection, suggest that this is a new human T-cell population that participates in immune response in TB., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Primary myelofibrosis is a myeloproliferative disorder characterized by bone marrow fibrosis, abnormal cytokine expression, splenomegaly and anemia. The activation of JAK2 and the increased levels of circulating proinflammatory cytokines seem to play an important role in the pathogenesis of myelofibrosis. Novel therapeutic agents targeting JAKs have been developed for the treatment of myeloproliferative disorders. Ruxolitinib (INCB018424) is the most recent among them., Case Presentation: To our knowledge, there is no evidence from clinical trials of an increased risk of tuberculosis during treatment with JAK inhibitors. Here we describe the first case of tuberculosis in a patient treated with Ruxolitinib, a male with a 12-year history of chronic idiopathic myelofibrosis admitted to our Institute because of fever, night sweats, weight loss and an enlarging mass in the left inguinal area for two months., Conclusion: Treatment with Ruxolitinib may have triggered the reactivation of latent tuberculosis because of an inhibition of Th1 response. Our case highlights the importance of an accurate screening for latent tuberculosis before starting an anti-JAK 2 treatment.

CD8 T-cells contribute to control of Mycobacterium tuberculosis infection, but little is known about the quality of the CD8 T-cell response in subjects with latent infection and in patients with active tuberculosis disease. CD8 T-cells recognizing epitopes from 6 different proteins of Mycobacterium tuberculosis were detected by tetramer staining. Intracellular cytokines staining for specific production of IFN-gamma and IL-2 was performed, complemented by phenotyping of memory markers on antigen-specific CD8 T-cells. The ex-vivo frequencies of tetramer-specific CD8 T-cells in tuberculous patients before therapy were lower than in subjects with latent infection, but increased at four months after therapy to comparable percentages detected in subjects with latent infection. The majority of CD8 T-cells from subjects with latent infection expressed a terminally-differentiated phenotype (CD45RA+CCR7(-)). In contrast, tuberculous patients had only 35% of antigen-specific CD8 T-cells expressing this phenotype, while containing higher proportions of cells with an effector memory- and a central memory-like phenotype, and which did not change significantly after therapy. CD8 T-cells from subjects with latent infection showed a codominance of IL-2+/IFN-gamma+ and IL-2(-)/IFN-gamma+ T-cell populations; interestingly, only the IL-2+/IFN-gamma+ population was reduced or absent in tuberculous patients, highly suggestive of a restricted functional profile of Mycobacterium tuberculosis-specific CD8 T-cells during active disease. These results suggest distinct Mycobacterium tuberculosis specific CD8 T-cell phenotypic and functional signatures between subjects which control infection (subjects with latent infection) and those who do not (patients with active disease).

Serum responses against Mycobacterium tuberculosis HSP16 were determined for children with tuberculosis (TB) and for healthy purified protein derivative (PPD)-positive and PPD-negative children. Immunoglobulin G (IgG) and IgM responses were higher for TB patients than for other groups. After chemotherapy, IgM and IgG responses decreased for TB patients and PPD-positive subjects. Monitoring of anti-M. tuberculosis HSP16 responses could assist in the management of pediatric TB.

Granulysin is a cytolytic protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Serum levels of granulysin are related to host cellular immunity. We used an ELISA to quantify granulysin serum levels in children with tuberculosis (TB), before and after chemotherapy. The study involved children affected by different clinical forms of TB (n=72) and healthy control children (n=150) from the same geographical area and of similar socio-economic background. Serum granulysin levels before the initiation of TB therapy were significantly lower in children with TB compared to controls, with the lowest levels being found in TB patients who were PPD skin test negative. No statistically significant differences were found between serum granulysin levels and clinical severity (mild/moderate or advanced pulmonary TB) or the clinical form (pulmonary or extra-pulmonary) of TB. At four months after completion of therapy, serum granulysin levels in children treated for TB were not significantly different to those observed in control children. This finding was paralleled by the increased in vitro mycobactericidal activity of sera from TB patients after completion of therapy. We propose that serum granulysin levels may provide a marker of disease activity in childhood TB and might be useful for monitoring improvement after chemotherapy.

In this study, we have examined the influence of HLA-DR molecules and the structure of the epitope repertoire of the 16-kDa protein of Mycobacterium tuberculosis on the acquisition of the cytokine secretion pattern of CD4 T cell clones, obtained from tuberculous patients before and after anti-mycobacterial therapy. Our data indicate that TB patients have a predominant Th0 response against the 16-kDa protein and its epitopes and that healing, induced by anti-mycobacterial therapy, is associated with a shift toward a predominant Th1 phenotype. Moreover, both HLA-DR molecules restricting the clone specificity and the nature of the recognized epitope do not play any role in the generation of Th0 and Th1 clones. These findings indicate that additional factors, such as the cytokine environment and/or costimulatory molecules, determine the Th phenotype of CD4 T cells during tuberculosis.

Tuberculosis (TB) kills 2 million people each year in the world, of which 250,000 are children. In Italy, paediatric TB is 3.5% of total cases with a steady trend in the last ten years. Childhood tuberculosis remains a disease of great concern because its occurrence always indicates recent transmission and is a pivotal indicator of effectiveness of TB control efforts. The epidemiological study, including DNA fingerprinting, of 71 children affected by TB - 62 pulmonary, 9 meningitis, 2 renal- shows that the source case is frequently a parent or household member. Sensitivity to anti-tubercular drugs was tested for 18/20 isolates obtained from the children and 21/44 isolates obtained from infection sources with 5 resistant strains in each group. One child was resistant to isoniazid, and one adult source to rifampin. Multi-drug resistance was observed in 8 cases: 4 children and 4 sources. In the children's case, we may use the term primary resistance as the patients have not been previously treated with the drugs. These children's treatment lasted longer, not only because their regimen had been changed, but also because of their delayed clinical-radiological response to the treatment. These data suggest that it would be opportune to re-evaluate current treatment of childhood tuberculosis, encouraging active and integrated cooperation between epidemiologists, infectious disease specialists and paediatricians.

Vgamma9/Vdelta2 T cells can contribute to protective immune response against Mycobacterium tuberculosis, although the extent to which and mechanisms by which they could actually protect against human tuberculosis remain unclear. We have previously reported that Vgamma9/Vdelta2 T cells from tuberculin purified protein derivative (PPD)-positive children, either healthy or affected by different clinical forms of tuberculosis, strongly proliferate to different phosphoantigens in vitro, whereas Vgamma9/Vdelta2 T cells from PPD-negative healthy subjects proliferate very poorly. We report here that Vgamma9/Vdelta2 T cells from tuberculous children have an increased proliferative activity, but decreased interferon (IFN)-gamma production and granulysin expression. After successful chemotherapy, the Vgamma9/Vdelta2 T cell proliferative response strongly decreased, whereas IFN-gamma and granulysin production consistently increased. Disease-associated changes in Vgamma9/Vdelta2 T cell effector functions in patients with tuberculosis are consistent with the possibility that these T cells may play a protective role in immune response against M. tuberculosis infection.

T cells mediate protection against tuberculosis, but little is known about their role during chemotherapy of patients with active disease. Here we examined the cytokine profile of CD4 T cells before and after four months of chemotherapy in six initial skin test anergic cases. Purified protein derivative (PPD) and 16-kDa antigen-reactive CD4 T-cell clones prior to therapy resided mostly in disease-associated body fluids and were of the Th0 (interferon (IFN)-gamma + interleukin (IL)-4) secreting profile. In contrast, the majority of postchemotherapy CD4 T-cell clones originated from blood and were of the IFN-gamma secreting Th1 type. However, the recognition of several peptides derived from the 16-kDa antigen was not significantly different between the Th1 and Th0 clones. We conclude that chemotherapy shifts CD4 T cells from the affected body fluids to the blood circulation, accompanied by a change from Th0 to Th1 cytokine profile.

The alphabeta and gammadelta T cell responses were analyzed in the peripheral blood of children affected by active tuberculosis (TB) and in healthy children who tested positive (PPD+) or negative (PPD-) for purified protein derivative. PPD+ healthy and diseased children responded equally well to PPD in vitro. In contrast, only 18% of PPD+ TB patients responded to peptide p38G derived from the 38-kDa protein of Mycobacterium tuberculosis. Analysis of the whole gammadelta T cell population and of its Vgamma9/Vdelta2 subset showed similar frequencies in PPD+ children with TB and in healthy PPD+ and PPD- children. Vgamma9/Vdelta2 cells from children with TB responded to 5 different phosphoantigens similarly to those from healthy PPD+ children, but healthy PPD- children responded very poorly. Chemotherapy had contrasting effects on the tested lymphocyte population, represented by increase of alphabeta and decline of Vgamma9/Vdelta2 T cell responses. T cell responses in childhood TB may be similar to those in adult TB.